Drosophila gastrulation: analysis of cell shape changes in living embryos by three-dimensional fluorescence microscopy.

The first event of Drosophila gastrulation is the formation of the ventral furrow. This process, which leads to the invagination of the mesoderm, is a classical example of epithelial folding. To understand better the cellular changes and dynamics of furrow formation, we examined living Drosophila embryos using three-dimensional time-lapse microscopy. By injecting fluorescent markers that visualize cell outlines and nuclei, we monitored changes in cell shapes and nuclear positions. We find that the ventral furrow invaginates in two phases. During the first 'preparatory' phase, many prospective furrow cells in apparently random positions gradually begin to change shape, but the curvature of the epithelium hardly changes. In the second phase, when a critical number of cells have begun to change shape, the furrow suddenly invaginates. Our results suggest that furrow formation does not result from an ordered wave of cell shape changes, contrary to a model for epithelial invagination in which a wave of apical contractions causes invagination. Instead, it appears that cells change their shape independently, in a stochastic manner, and the sum of these individual changes alters the curvature of the whole epithelium.     

A biomechanical analysis of ventral furrow formation in the Drosophila melanogaster embryo

The article provides a biomechanical analysis of ventral furrow formation in the Drosophila melanogaster embryo. Ventral furrow formation is the first large-scale morphogenetic movement in the fly embryo. It involves deformation of a uniform cellular monolayer formed following cellularisation, and has therefore long been used as a simple system in which to explore the role of mechanics in force generation. Here we use a quantitative framework to carry out a systematic perturbation analysis to determine the role of each of the active forces observed. The analysis confirms that ventral furrow invagination arises from a combination of apical constriction and apical-basal shortening forces in the mesoderm, together with a combination of ectodermal forces. We show that the mesodermal forces are crucial for invagination: the loss of apical constriction leads to a loss of the furrow, while the mesodermal radial shortening forces are the primary cause of the internalisation of the future mesoderm as the furrow rises. Ectodermal forces play a minor but significant role in furrow formation: without ectodermal forces the furrow is slower to form, does not close properly and has an aberrant morphology. Nevertheless, despite changes in the active mesodermal and ectodermal forces lead to changes in the timing and extent of furrow, invagination is eventually achieved in most cases, implying that the system is robust to perturbation and therefore over-determined.     

Gastrulation in Drosophila

The three germ layers in Drosophila are established by both the invagination of the ventral furrow, which internalizes the anterior midgut and mesoderm primordia, and the invagination of the posterior midgut primordium. The invaginations of these primordia occur by similar cell shape changes. The gene hierarchies responsible for positioning each primordium within the epithelial blastoderm are well understood. By going further down in the hierarchy, we hope to identify the genes whose products are directly involved in the mechanisms that change the cell shape. Presumably these mechanisms are similar in Drosophila and in other organisms.     

Mechanical feedback and robustness of apical constrictions in Drosophila embryo ventral furrow formation

Formation of the ventral furrow in the Drosophila embryo relies on the apical constriction of cells in the ventral region to produce bending forces that drive tissue invagination. In our recent paper we observed that apical constrictions during the initial phase of ventral furrow formation produce elongated patterns of cellular constriction chains prior to invagination and argued that these are indicative of tensile stress feedback. Here, we quantitatively analyze the constriction patterns preceding ventral furrow formation and find that they are consistent with the predictions of our active-granular-fluid model of a monolayer of mechanically coupled stress-sensitive constricting particles. Our model shows that tensile feedback causes constriction chains to develop along underlying precursor tensile stress chains that gradually strengthen with subsequent cellular constrictions. As seen in both our model and available optogenetic experiments, this mechanism allows constriction chains to penetrate or circumvent zones of reduced cell contractility, thus increasing the robustness of ventral furrow formation to spatial variation of cell contractility by rescuing cellular constrictions in the disrupted regions.     

Invagination of the otic placode: normal development and experimental manipulation

The inner ear forms from paired ectodermal primordia that lie to either side of the developing hindbrain. Initially each primordium forms a shallow depression in the ectodermal surface. Invagination to form an otic pit coincides with the formation of several deep folds in the epithelial surface. An initial fold appears parallel to the embryonic axis and at the junction of the rhombencephalon with somitomeric mesoderm. This is followed by formation of cranial and caudal folds perpendicular to the axis and minor folds that are within the pit formed by earlier folding. The central region of the otic primordium remains in close apposition to the lateral surface of the neural tube during the process of fold formation, until the otic pit becomes quite deep. At that time, mesenchymal cells penetrate between the two layers. Experimental analysis of invagination supports the conclusion that otic invagination is controlled differently from that of similar organ primordia, such as the eye and thyroid. Whereas these other primordia can be stimulated to undergo normal morphogenetic shape changes precociously by treatments that presumably activate motile processes in the cytoskeleton, the same conditions have little effect on the otic placode. Similarly, neither inhibitors of calcium transport nor inactivators of calmodulin activity prevent otic pit formation, while these drugs block invagination of other primordia. These results suggest that otic invagination may be caused by changes in the surrounding tissues rather than by an activation of motility within the primordium.     

Stress-dependent morphogenesis: continuum mechanics and truss systems

A set of equilibrium equations is derived for the stress-controlled shape change of cells due to the remodelling and growth of their internal architecture. The approach involves the decomposition of the deformation gradient into an active and a passive component; the former is allowed to include a growth process, while the latter is assumed to be hyperelastic and mass-preserving. The two components are coupled with a control function that provides the required feedback mechanism. The balance equations for general continua are derived and, using a variational approach, we deduce the equilibrium equations and study the effects of the control function on these equations. The results are applied to a truss system whose function is to simulate the cytoskeletal network constituted by myosin microfilaments and microtubules, which are found experimentally to control shape change in cells. Special attention is paid to the conditions that a thermodynamically consistent formulation should satisfy. The model is used to simulate the multicellular shape changes observed during ventral furrow invagination of the Drosophila melanogaster embryo. The results confirm that ventral furrow invagination can be achieved through stress control alone, without the need for other regulatory or signalling mechanisms. The model also reveals that the yolk plays a distinct role in the process, which is different to its role during invagination with externally imposed strains. In stress control, the incompressibility constraint of the yolk leads, via feedback, to the generation of a pressure in the ventral zone of the epithelium that eventually eases its rise and internalisation.     

Neural cell fate in rca1 and cycA mutants: the roles of intrinsic and extrinsic factors in asymmetric division in the Drosophila central nervous system.

In the central nervous system (CNS) of Drosophila embryos lacking regulator of cyclin A (rca1) or cyclin A, we observe that several ganglion mother cells (GMCs) fail to divide. Whereas GMCs normally produce two sibling neurons that acquire different fates ('A/B'), non-dividing GMCs differentiate exclusively in the manner of one of their progeny ('B'). In zygotic numb mutants, sibling neuron fate alterations ('A/B' to 'A/A') occur infrequently or do not occur in some sibling pairs; we have determined that depletion of both maternal and zygotic numb causes sibling neurons to acquire equalized fates ('A/A') with near-complete expressivity. In rca1, numb mutant embryos, we observe binary cell fate changes ('B' to 'A') in several GMCs as well. Finally, we have demonstrated that expression of Delta in the mesoderm is sufficient to attain both sibling fates. Our results indicate that the intrinsic determinant Numb is absolutely required to attain differential sibling neuron fates. While the extrinsic factors Notch and Delta are also required to attain both fates, our results indicate that Delta signal can be received from outside the sibling pair.     

Gastrulation in Drosophila: the formation of the ventral furrow and posterior midgut invaginations

The ventral furrow and posterior midgut invaginations bring mesodermal and endodermal precursor cells into the interior of the Drosophila embryo during gastrulation. Both invaginations proceed through a similar sequence of rapid cell shape changes, which include apical flattening, constriction of the apical diameter, cell elongation and subsequent shortening. Based on the time course of apical constriction in the ventral furrow and posterior midgut, we identify two phases in this process: first, a slow stochastic phase in which some individual cells begin to constrict and, second, a rapid phase in which the remaining unconstricted cells constrict. Mutations in the concertina or folded gastrulation genes appear to block the transition to the second phase in both the ventral furrow and the posterior midgut invaginations.     

A Preferred Curvature-Based Continuum Mechanics Framework for Modeling Embryogenesis

Mechanics plays a key role in the development of higher organisms. However, understanding this relationship is complicated by the difficulty of modeling the link between local forces generated at the subcellular level and deformations observed at the tissue and whole-embryo levels. Here we propose an approach first developed for lipid bilayers and cell membranes, in which force-generation by cytoskeletal elements enters a continuum mechanics formulation for the full system in the form of local changes in preferred curvature. This allows us to express and solve the system using only tissue strains. Locations of preferred curvature are simply related to products of gene expression. A solution, in that context, means relaxing the system’s mechanical energy to yield global morphogenetic predictions that accommodate a tendency toward the local preferred curvature, without a need to explicitly model force-generation mechanisms at the molecular level. Our computational framework, which we call SPHARM-MECH, extends a 3D spherical harmonics parameterization known as SPHARM to combine this level of abstraction with a sparse shape representation. The integration of these two principles allows computer simulations to be performed in three dimensions on highly complex shapes, gene expression patterns, and mechanical constraints. We demonstrate our approach by modeling mesoderm invagination in the fruit-fly embryo, where local forces generated by the acto-myosin meshwork in the region of the future mesoderm lead to formation of a ventral tissue fold. The process is accompanied by substantial changes in cell shape and long-range cell movements. Applying SPHARM-MECH to whole-embryo live imaging data acquired with light-sheet microscopy reveals significant correlation between calculated and observed tissue movements. Our analysis predicts the observed cell shape anisotropy on the ventral side of the embryo and suggests an active mechanical role of mesoderm invagination in supporting the onset of germ-band extension.     

Expression patterns of twist and snail in Tribolium (Coleoptera) suggest a homologous formation of mesoderm in long and short germ band insects

The mesodermal region in Drosophila is determined by a maternally derived morphogenetic gradient system which specifies the different cell fates along the dorsoventral axis, including the prospective mesodermal cells at the ventral side of the embryo. There are at least two zygotic target genes, twist and snail, which are required for mesoderm formation in Drosophila. To analyze whether a similar mode of mesoderm specification might also apply to short germ band insect embryos, we have cloned twist and snail- related gene fragments from the flour beetle Tri-bolium and have analyzed their expression pattern. Both genes are expressed in a ventral stripe at early blastoderm stage, which is restricted to the region of the developing germ rudiment. The cells expressing the two genes are those that invaginate during gastrulation, indicating that the early stages of mesoderm specification are indeed very similar between the two species. Interestingly, both genes are also expressed during germband extension in a subregion of the growth zone of the embryo which forms the mesodermal cells. This suggests that the expression of the two genes is required for mesoderm formation both at early blastoderm stage and during germband elongation until the end of the segmental growth process. © 1994 Wiley-Liss, Inc.     

Passive Mechanical Forces Control Cell-Shape Change during Drosophila Ventral Furrow Formation

During Drosophila gastrulation, the ventral mesodermal cells constrict their apices, undergo a series of coordinated cell-shape changes to form a ventral furrow (VF) and are subsequently internalized. Although it has been well documented that apical constriction is necessary for VF formation, the mechanism by which apical constriction transmits forces throughout the bulk tissue of the cell remains poorly understood. In this work, we develop a computational vertex model to investigate the role of the passive mechanical properties of the cellular blastoderm during gastrulation. We introduce to our knowledge novel data that confirm that the volume of apically constricting cells is conserved throughout the entire course of invagination. We show that maintenance of this constant volume is sufficient to generate invagination as a passive response to apical constriction when it is combined with region-specific elasticities in the membranes surrounding individual cells. We find that the specific sequence of cell-shape changes during VF formation is critically controlled by the stiffness of the lateral and basal membrane surfaces. In particular, our model demonstrates that a transition in basal rigidity is sufficient to drive VF formation along the same sequence of cell-shape change that we observed in the actual embryo, with no active force generation required other than apical constriction.     

Passive Mechanical Forces Control Cell-Shape Change during Drosophila Ventral Furrow Formation

During Drosophila gastrulation, the ventral mesodermal cells constrict their apices, undergo a series of coordinated cell-shape changes to form a ventral furrow (VF) and are subsequently internalized. Although it has been well documented that apical constriction is necessary for VF formation, the mechanism by which apical constriction transmits forces throughout the bulk tissue of the cell remains poorly understood. In this work, we develop a computational vertex model to investigate the role of the passive mechanical properties of the cellular blastoderm during gastrulation. We introduce to our knowledge novel data that confirm that the volume of apically constricting cells is conserved throughout the entire course of invagination. We show that maintenance of this constant volume is sufficient to generate invagination as a passive response to apical constriction when it is combined with region-specific elasticities in the membranes surrounding individual cells. We find that the specific sequence of cell-shape changes during VF formation is critically controlled by the stiffness of the lateral and basal membrane surfaces. In particular, our model demonstrates that a transition in basal rigidity is sufficient to drive VF formation along the same sequence of cell-shape change that we observed in the actual embryo, with no active force generation required other than apical constriction.     

Ingression of primary mesenchyme cells of the sea urchin embryo: a precisely timed epithelial mesenchymal transition

Epithelial-mesenchyme transitions (EMTs) are familiar to all scholars of development. Each animal system utilizes an EMT to produce mesenchyme cells. In vertebrates, for example, there are a number of EMTs that shape the embryo. Early, entry of epiblast cells into the primitive streak is followed by the emergence of mesoderm via an EMT process. The departure of neural crest cells from the margin of the neural folds is an EMT process, and the delamination of cells from the endomesoderm to form the supporting mesenchyme of the lung, liver, and pancreas are EMTs. EMTs are observed in Drosophila following invagination of the ventral furrow, and even in Cnidarians, which have only two germ layers, yet mesoglial and stem cells delaminate from the epithelia and occupy the matrix between the ectoderm and endoderm. This review will focus on a classic example of an EMT, which occurs in the sea urchin embryo. The primary mesenchyme cells (PMCs) ingress from the vegetal plate of this embryo precociously and in advance of archenteron invagination. Because ingression is precisely timed, the PMC lineage precisely known, and the embryo easily observed and manipulated, much has been learned about how the ingression of PMCs works in the sea urchin. Though the focus of this review is the sea urchin PMCs, there is evidence that all EMTs share many common features at both cellular and molecular levels, and many of these mechanisms are also shown to be involved in tumor progression, especially metastasizing carcinomas.